1. Field of the Invention
The subject-manner of the invention is a strain for production of a live, orally applicable Escherichia coli vaccine for the prevention of post-weaning diarrhoea in pigs, and the procedure suitable for producing that strain.
2. Description of Related Art
Diarrhoea caused by so-called enterotoxigenic Escherichia coli (ETEC) bacteria is one of the most severe, commonly occurring diseases of pig rearing, which usually causes 5–15% mortality in newborn (baby) piglets, and subsequently gives rise to losses of similar magnitude after weaning. Losses arising from the costs of medication, growth retardation and other consequences of the disease are much more substantial than direct damage due to mortality. The pathogenesis of the disease is essentially as follows: after getting into the intestinal tract of pigs, ETEC bacteria adhere to the wall of the small intestine with the help of their surface protein antigens (fimbriae), multiply there in large numbers, and transfer their heat-stable or heat-labile toxins (STa, STb, LT enterotoxins) directly to the intestinal epithelial cells. Due to the effect exerted by the toxins, the fluid-absorbing activity of intestinal epithelial cells will cease and the cells will secrete a large volume of fluid into the intestinal lumen. As a result, clinically severe diarrhoea will develop. Thus, the first step in the pathogenesis of the disease entity is the adherence of pathogenic ETEC bacteria to the intestinal wall (which adherence itself does not cause disease), followed by the effect exerted by the toxins on the intestinal epithelial cells (this results in diarrhoea). [Fairbrother, J M.: Enteric colibacillosis. In: Leman A D, Straw B E, Mengeling W L, D'Allaire S, Taylor D, eds. Diseases of Swine, 7th edn, Ames, Iowa state University Press, 1992: 489–497.]
In the past 10–15 years, several vaccines have been developed and marketed for the control of neonatal coli diarrhea. Through vaccination of the dams (sows) (so-called parenteral vaccination) these vaccines induce the production of specific antibodies to pathogenic ETEC strains in the sow's milk. These antibodies will provide newborn and baby piglets with adequate protection. The antibodies present in the mother's milk specifically inhibit the growth of pathogens located in the intestinal lumen and on the intestinal wall. [Moon H W.: Protection against enteric colibacillosis in pigs suckling orally vaccinated dams: Evidence for pili as protective antigens. Am. J. Vet. Res. 1981: 42, 173–177.]
For the control of post-weaning coli diarrhoea of piglets, no effective vaccine is known yet which could be used in a manner similar to that described above. This is mostly due to the fact that vaccines administered parenterally (i.e. not through the alimentary canal) predominantly facilitate the production of circulating antibodies which, however, can hardly, or not at all, reach the enterotoxic Escherichia coli bacteria located in the intestinal lumen and on the intestinal wall. Therefore, certain researchers attempt to administer the antibodies itself (e.g. specific antibodies produced in the yolk of chicken eggs) into the gastrointestinal tract of piglets by the oral route (e.g mixed in the feed) [Zuniga A, Yokohama H, Albicker-Rippinger P, Eggenberger E, Bertschinger H U.: Reduced intestinal colonization with F18-positive enterotoxigenic Escherichia coli in weaned pigs fed chicken egg antibody against the fimbriae. FEMS Immunol. Medical Microbiol. 1997: 18, 153–161]. A further possible method of control is the use of orally applicable vaccines that exert their immunizing effect against ETEC bacteria in the digestive tract. Earlier vaccines of this type, suitable for immunizing the intestinal tract, contained inactivated microorganisms. In certain cases the continuous presence of such microorganisms in substantial amounts (in the feed) reduced the losses caused by post-weaning diarrhoea; however, nowadays such vaccines are used rarely or not at all due to their low reliability. In this case one of the main causes of failure is that heat treatment destroys those heat-labile, proteinaceous products of ETEC bacteria which are important for their virulence. These are the heat-labile enterotoxin, and the F4 [Orskov I, Orskov F.: Serology of Escherichia coli fimbriae. Prog. Allergy 1983: 33, 80–105.] and F18 [Rippinger P, Bertschinger H U, Imberechts H, Nagy B, Sorg I, Stamm M, Wild P, Wittig W.: Designations F18ab, and F18ac for the related fimbrial types F107, 2134P and 8813 of Escherichia coli isolated from porcine postweaning diarrhoea and from oedema disease. Vet. Microbiol. 1995: 45, 281–295.] fimbriae. Thus, they cannot exert their immunizing effect. As a consequence, such vaccines—to be produced primarily for immunizing the intestinal tract—must contain live microorganisms that can colonize the intestinal tract, can produce there the antigens important for protection continuously and in sufficient quantity, and can facilitate the immunity of the intestinal tract against them.
Live microorganisms capable of providing local protection (i.e. protection asserting itself on the mucous membrane) against pathogens can be developed either by inserting genes into innocuous laboratory strains or by altering certain properties of the pathogens. We chose the latter approach.